Treating and preventing disease with tma  and tmao lowering agents

ABSTRACT

Provided herein are compositions, systems, and methods for treating a disease, such as kidney and/or cardiovascular disease, with an agent that reduces the production of trimethylamine (TMA) or trimethylamine-n-oxide (TMAO) in a subject. In certain embodiments, the agent is: i) 3,3-dimethyl-1-butanol (DMB) or a DMB derivative or related compound, ii) acetylsalicylic acid or derivative thereof (e.g., with an enteric coating for delivery to the colon and/or cecum); iii) a flavin monooxygenase 3 (FMO3) inhibitor; iv) a gut TMA lyase inhibitor; v) an antibiotic or antimicrobial; vi) a probiotic or prebiotic; vii) an antiplatelet agent; or viii) a TMA and/or TMAO sequestering agent.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present invention is a continuation of U.S. patent application Ser.No. 16/159,102, filed Oct. 12, 2018, which is a continuation of U.S.patent application Ser. No. 15/610,145, filed May 31, 2017, now U.S.Pat. No. 10,117,879, which is a continuation of U.S. patent applicationSer. No. 14/866,398, filed Sep. 25, 2015, now U.S. Pat. No. 9,694,020,which claims priority to U.S. Provisional Patent Application 62/056,168,filed Sep. 26, 2014, each of which is incorporated by reference in itsentirety.

FIELD OF THE INVENTION

Provided herein are compositions, systems, and methods for treating adisease, such as kidney disease and/or cardiovascular disease, with anagent that reduces the production of trimethylamine (TMA) ortrimethylamine-n-oxide (TMAO) in a subject. In certain embodiments, theagent is: i) 3,3-dimethyl-1-butanol (DMB) or a DMB derivative or relatedcompound, ii) acetylsalicylic acid or derivative thereof (e.g., with anenteric coating for delivery to the colon and/or cecum); iii) a flavinmonooxygenase 3 (FMO3) inhibitor; iv) a gut TMA lyase inhibitor; v) anantibiotic or antimicrobial; vi) a probiotic or prebiotic; vii) anantiplatelet agent, or vii) a TMA and/or TMAO sequestering agent.

BACKGROUND

Kidneys of the human body function to remove excess fluids as well assome ions. The functional unit of the kidney is the nephron. A nephronconsists of a filtering unit of tiny blood vessels called a glomerulusattached to a tubule. When blood enters the glomerulus, it is filteredand the remaining fluid then passes along the tubule. In the tubule,chemicals and water are either added to or removed from this filteredfluid according to the body's needs, and the final product is urine,which is excreted.

In patients with chronic kidney disease, kidney function is severelycompromised. Chronic kidney disease (CKD), also known as chronic renaldisease, is a progressive loss in renal function over a period of monthsor years. The most severe stage of CKD is End Stage Renal Disease(ESRD), which occurs when the kidneys cease to function. The two maincauses of CKD are diabetes and high blood pressure, which areresponsible for up to two-thirds of the cases. Heart disease is theleading cause of death for all people having CKD. Excessive fluid canaccumulate in patients suffering from ESRD. The mortality rate of ESRDpatients who receive traditional hemodialysis therapy is 24% per yearwith an even higher mortality rate among diabetic patients. Fluidaccumulates in ESRD patients because the kidneys can no longereffectively remove water and other fluids from the body. The fluidaccumulates first in the blood and then accumulates throughout the body,resulting in swelling of the extremities and other tissues as edema.This accumulation of fluid causes increased stress on the heart, in turncausing significant increases in blood pressure or hypertension, whichcan lead to heart failure.

Although the population of patients afflicted with CKD grows each year,there is no cure. Current treatments for CKD seek to slow theprogression of the disease. However, as the disease progresses, renalfunction decreases, and, eventually, renal replacement therapy isemployed to compensate for lost kidney function. Renal replacementtherapy entails either transplantation of a new kidney or dialysis.

Methods to treat kidney disease require the processing of blood toextract waste components such as urea and ions. The traditionaltreatment for kidney disease involves dialysis. Dialysis emulates kidneyfunction by removing waste components and excess fluid from a patient'sblood. This is accomplished by allowing the body fluids, usually theblood, to come into the close proximity with the dialysate, which is afluid that serves to cleanse the blood and actively remove the wastecomponents and excess water. During this process, the blood anddialysate are separated by a dialysis membrane, which is permeable towater, small molecules (such as urea), and ions but not permeable to thecells. Each dialysis session lasts a few hours and may be repeated asoften as three times a week.

Traditional processes, such as dialysis, require extracorporealprocessing of body fluids. Once the blood is purified, it is thenreturned to the patient. Although effective at removing waste componentsfrom blood, dialysis treatments are administered intermittently and,therefore, do not emulate the continuous function of a natural kidney.Once the dialysis session is completed, the fluid begins to accumulateagain in the tissues of the patient. The benefits of dialysisnotwithstanding, statistics indicate that three out of five dialysispatients die within five years of commencing treatment. Studies haveshown that increasing the frequency and duration of dialysis sessionscan improve the survivability of dialysis patients. Increasing thefrequency and duration of dialysis sessions more closely resembles thecontinuous kidney function sought to be emulated. However, theextracorporeal processing of the body fluids increases the discomfort,inconvenience and the costs associated with treatment. There is also anadditional risk of infection, which mandates that the procedures becarried out under the supervision of trained medical personnel.

SUMMARY OF THE INVENTION

Provided herein are compositions, systems, and methods for treating adisease, such as kidney disease, or cardiovascular disease, with anagent that reduces the production of trimethylamine (TMA) ortrimethylamine-n-oxide (TMAO) in a subject. In certain embodiments, theagent is: i) 3,3-dimethyl-1-butanol (DMB) or a DMB derivative or relatedcompound, ii) acetylsalicylic acid or derivative thereof (e.g., with anenteric coating for delivery to the colon and/or cecum); iii) a flavinmonooxygenase 3 (FMO3) inhibitor; iv) a gut TMA lyase inhibitor; v) anantibiotic or antimicrobial; vi) a probiotic or prebiotic; vii) anantiplatelet agent; vii) a TMA and/or TMAO sequestering agent.

In some embodiments, provided herein are methods of treating orpreventing kidney disease, and/or cardiovascular disease, comprising:treating a subject with an agent or procedure (or prescribing orrecommending treating with an agent or procedure), wherein: i) thesubject has symptoms of kidney disease, and/or cardiovascular disease,and the treating reduces or eliminates at least one symptom of thekidney and/or cardiovascular disease; ii) the subject is apparentlyhealthy, but has elevated levels of TMAO, and the treating prevents thedevelopment of chronic kidney and/or cardiovascular disease in thesubject; or iii) the subject has age-related decline in kidney function,and the treating prevents or attenuates further decline in the kidneyfunction; and iv) said the or procedure is selected from: a)3,3-dimethyl-1-butanol (DMB) or a DMB derivative or related compound(e.g., as shown in Table 3); b) acetylsalicylic acid with or without anenteric coating; c) an acetylsalicylic acid derivative with or withoutan enteric coating; d) a flavin monooxygenase 3 (FMO3) inhibitor; e) agut TMA lyase inhibitor; f) fecal microbiota transplantation; g)delivery of acetylsalicylic acid or derivative thereof directly to thecolon or cecum of the subject; h) an antibiotic or antimicrobial thatreduces trimethylamine (TMA) production in the gut; i) a probiotic orprebiotic that reduces TMA production in the gut; j) an antiplateletagent; and k) a TMA and/or TMAO sequestering agent. In certainembodiments, a subject is treated prophylactically with the agents andprocedures mentioned herein to promote renal health and prevent TMAOinduced impairment of renal function.

In particular embodiments, rather than, or in addition to treating, thepatient is prescribed one of the agents or procedures described herein.In some embodiments, a patient with kidney and/or cardiovascular diseaseis also, or alternatively, prescribed a diet with reduced levels ofcarnitine containing compounds (e.g., prescribed a vegetarian or vegandiet). In certain embodiments, the diet is a Mediterranean diet, or dietlow in TMAO precursors (e.g., low in choline, lecithin, carnitine,etc.), or low in TMAO (e.g., a diet low in certain fish, such as cod,tilapia, Chilean sea bass, etc.).

In certain embodiments, a sample from the subject is assayed todetermine levels of eGFR, eCrCl, Cystatin C, KIM1, microalbuniuria (anelevated urine albumin/Creatinine ratio), trimethylamine N-oxide (TMAO),TMA, and/or a TMA-containing compound prior to and/or after saidtreating. In particular embodiments, a subject is found to needtreatment if elevated levels of KIM1, TMA, TMAO, or elevated urinealbumin/Creatine ratio are found. In other embodiments, a subject isfound to need treatment if decreased levels of eFGF, eCrCl, or increasedCystatin C are found. In other embodiments, the sample comprises wholeblood, serum, plasma, exhaled breath, urine, saliva, cerebrospinalfluid, or bronchoalveolar lavage.

In other embodiments, the DMB derivative or related compound is as shownin Formula I below:

-   wherein n is an integer, or n is 0, indicating that CH₂ is not    present;-   wherein each W is independently selected from: H, Cl, F, Br, or I    (e.g., W₃C═CH₃, CH₂Cl, CH₂Fl, CH₂Br, CH₂I, CF₃, CCl₃, CBr₃, CI₃, or    CHCl₂);-   wherein Y is C, N, Si, P, S, Ge, Sn, Pb, P, As, Sb, or Bi;-   wherein X is O or S and the corresponding bond is either present or    absent or double,-   wherein R is absent, H, an alkyl group, alkenyl group, alkynyl    group, phenyl group, amide, alkylamide, or a benzyl group;-   wherein Z is C, CH₂, CH, NH, O, or S,-   wherein XR is, alternatively, H, an ester, thioester, or thionester;    glycerol, or one of the following three formulas:

-   wherein R′ is H, an alkyl group, alkenyl group, alkynyl group,    phenyl group, or a benzyl group; and wherein X′ is O, or S. In    certain embodiments, R is amide or alkylamide, and Z is an O, and Z    as a double bond O—a carboxylic acid). In some embodiments, the two    methyl groups extending from Y are linked by an alkyl or ether to    form a 4-6 member ring.

In further embodiments, the acetylsalicylic acid derivative or relatedcompound is selected from the group consisting of: 4-Methylsalicylicacid, 5-(acetylamino)-2-hydroxybenzoic acid; Salicylic acid, sodiumsalt, 4-Aminosalicylic acid, 3-Methylsalicylic acid, 3-Nitrosalicylicacid, 1-Hydroxy-2-naphthoic acid, 2-Hydroxyethyl salicylate,5-Bromosalicylic acid, 5-Methylsalicylic acid, 5-Aminosalicylic acid,2,4-Dihydroxybenzoic acid, 2,4-Dimethoxybenzoic acid,3-Hydroxy-2-naphthoic acid, 5-Nitrosalicylic acid, Phenyl salicylate,Ethyl salicylate, 5-Iodosalicylic acid, Methyl salicylate,5,5′-Methylenedisalicylic acid, Pamoic acid, 2-Ethoxybenzoic acid,2,6-Dihydroxybenzoic acid, 2,3-Dihydroxybenzoic acid, Ochratoxin A,5-Chlorosalicyclic acid, 4-Fluorosalicylic acid, Methyl5-fluoro-2-hydroxybenzoate, 2,4,5-Trimethoxybenzoic acid,2,5-Dihydroxybenzoic acid, Acetylsalicylsalicylic acid, Salicylsalicylicacid, 6-Methylsalicylic acid, Aluminon, 3-Aminosalicylic acid,2,3,4-Trimethoxybenzoic acid, o-Anisic acid, Isopropyl salicylate,3,5-Dinitrosalicylic acid, 2,3,4-Trihydroxybenzoic acid,5-Formylsalicylic acid, 2-Hydroxy-4-nitrobenzoic acid, Lithium3,5-diiodosalicylate, 4-Fluorosulfonyl-1-hydroxy-2-naphthoic acid,3-Methoxysalicylic acid, Methyl 1-hydroxy-2-naphthoate, Carminic acid,Carmine (pure, alum lake of carminic acid), Carmine (high puritybiol.stain, alum lake of carminic acid), 2,6-Dimethoxybenzoic acid,2,3-Dimethoxybenzoic acid, Chrome Azurol S, Alizarin Yellow R sodiumsalt, 3-Chlorosalicylic acid, 2-(trifuloromethoxy)benzoic acid, Methyl2,4-dimethoxybenzoate, Methyl 2,6-dihydroxybenzoate, Methyl2,4-dihydroxybenzoate, Triethanolamine salicylate, 2-Ethoxynaphthoicacid, 4-Methoxysalicylic acid, 5-Methoxysalicylic acid,2,5-Dimethoxybenzoic acid, 3,5 Dibromosalicylic acid, 6-Methoxysalicylicacid, 5-Chloro-o-anisic acid, Chromoxane Cyanine R,3-Hydroxy-4-(2-hydroxy-4-sulfo-1-naphthylazo)naphthalene-2-carboxylicacid, indicator grade ethyl 2,3-dihydroxybenzoate, Methyl5-iodosalicylate, methyl 5-chloro-2-hydroxybenzoate, Methyl4-acetamido-5-chloro-2-methoxybenzoate, 2-(acetyloxy)-3-methylbenzoicacid, 2-(acetyloxy)-3-methylbenzoic acid, 1,4-Benzodioxan-5-carboxylicacid, 2-Methoxy-5-(trifluoromethyl)benzoic acid, 4-Chlorosalicylic acid,Methyl 4-methoxysalicylate, 1,3-benzodioxole-4-carboxylic acid,5-Sulfosalicylic acid dihydrate, 5-Sulfosalicylic acid dihydrate,5-Sulfosalicylic acid dihydrate, Mordant Yellow 10,4-Amino-5-chloro-2-methoxybenzoic acid, Methyl 5-acetylsalicylate,5-chlorosulfonyl-2-hydroxybenzoic acid, methyl2-[2-(dimethylamino)ethoxy]benzoate, alpha-Apo-oxytetracycline,beta-Apo-oxytetracycline, 3,5-Di-tert-butylsalicylic acid, Methyl3,5-dibromo-2-hydroxybenzoate, 2-(3-methoxyphenoxy)benzoic acid, Methyl3-nitrosalicylate, Methyl 5-methyl salicylate, methyl4-amino-2-methoxybenzoate, chroman-8-carboxylic acid, methyl2,5-di(2,2,2-trifluoroethoxy)benzoate, 2,3dihydrobenzo[b]furan-7-carboxylic acid, methyl3-amino-2-hydroxybenzoate, 3-chloro-2,6-dimethoxybenzoic acid,3-Hydroxyphthalic anhydride,5-Bromo-2,3-dihydrobenzo[b]furan-7-carboxylic Acid,2,2-dimethyl-2,3-dihydro-1-benzofuran-7-carboxylic acid,6-Fluorosalicylic acid, 2,4,6-Trihydroxybenzoic acid monohydrate,3-bromo-2,6-dimethoxybenzoic acid, 3-bromo-2,6-dimethoxybenzoic acid,3,5-dichloro-2,6-dimethoxybenzoic acid, Lavendustin A,2-Fluoro-6-methoxybenzoic acid, 5-Bromo-2,4-dihydroxybenzoic acidmonohydrate, 3-chloro-2,6-dimethoxy-5-nitrobenzoic acid, methyl4,7-dibromo-3-methoxy-2-naphthoate, 2-(trifluoromethoxy)terephthalicacid, 2-methoxy-4,6-di(trifluoromethyl)benzoic acid,2-[2-(dimethylamino)ethoxy]benzoic acid,2-[(5-chloro-3-pyridyl)oxy]-5-nitrobenzoic acid,6-fluoro-4H-1,3-benzodioxine-8-carboxylic acid,3-Methoxy-4-(methoxycarbonyl)phenylboronic acid pinacol ester,3-Methoxy-4-(methoxycarbonyl)phenylboronic acid,2-(tetrahydropyran-4-yloxy)benzoic acid, pentafluorophenyl2-(tetrahydro-2H-pyran-4-yloxy)benzoate,3-Hydroxy-4-(methoxycarbonyl)phenylboronic acid pinacol ester, and3-Formylsalicylic acid hydrate.

In particular embodiments, the FMO3 inhibitor comprises Tenofovir orMethimazole. In some embodiments, the antibiotic is a broad spectrumantibiotic. In further embodiments, the antibiotic is one antibiotic ora combination of antibiotics selected from the group consisting of:metronidazole, ciprofloxacin, and neomycin, amoxicillin. In particularembodiments, the antiplatelet agent is selected from the groupconsisting of: abciximab, dipyridamole/ASA, anagrelide, cilostazol,clopidogrel, dipyridamole, eptifabatide, prasugrel, ticagrelor,ticlopidine, tirofiban, and vorapaxar. In further embodiments, theenteric coating provides for release of a majority of theacetylsalicylic acid or the acetylsalicylic acid derivative in the colonor cecum of the subject.

In some embodiments, the TMA and/or TMAO sequestering agent comprisesactivated charcoal or copper chlorophyllin (e.g., activated charcoal at750 mg 2×/day for 10 days, or copper chlorophyllin at 60 mg 3×/day aftermeals for 3 weeks).

In some embodiments, provided herein are systems comprising: a) a reportfor a patient with kidney and/or cardiovascular disease indicating thatthe patient has elevated levels of TMA or TMAO; and b) an agent fortreating kidney and/or cardiovascular disease selected from: i)3,3-dimethyl-1-butanol (DMB) or a DMB derivative or related compound(e.g., as shown in Table 3); ii) acetylsalicylic acid (aspirin) with orwithout an enteric coating; iii) an acetylsalicylic acid derivative withor without an enteric coating; iv) a flavin monooxygenase 3 (FMO3)inhibitor; v) a gut TMA lyase inhibitor; vi) fecal microbiotatransplantation agents; vii) an antibiotic or antimicrobial that reducesTMA production in the gut; viii) a probiotic or prebiotic that reducesTMA production in the gut; ix) an antiplatelet agent, and x) a TMAand/or TMAO sequestering agent.

In certain embodiments, provided herein are methods for treating adisease associated with elevated TMA and/or trimethylamine N-oxide (TMAOlevels) comprising: treating a subject with symptoms of a diseaseassociated with elevated TMA and/or TMAO levels with an agent comprisingacetylsalicylic acid or an acetylsalicylic acid derivative, wherein thetreating is under condition such that a majority or all of the agent isdelivered to the subject's colon and/or cecum, and wherein the treatingresults in at least one symptom of the disease being reduced oreliminated.

In certain embodiments, the disease is a cardiovascular disease (e.g.,Coronary Heart Disease, Cor Pulmonale, Congenital Heart Defect,Cardiomyopathy, Myocardial Infarction, Congestive Heart Failure,Valvular Heart Disease, Arrhythmia, Peripheral Arterial Disease,Cerebrovascular Accident, and Rheumatic Heart Disease). In certainembodiments, the disease is kidney disease. In other embodiments, theagent comprises an enteric coating that provides for release of amajority of the agent in the colon and/or cecum. In further embodiments,a sample from the subject is assayed to determine levels oftrimethylamine N-oxide (TMAO), TMA, and/or a TMA-containing compoundprior to and/or after the treating.

In some embodiments, provided herein are systems comprising: a) a reportfor a patient with a disease associated with elevated levels of TMAOand/or TMA indicating that the patient has elevated levels of TMAOand/or TMA; and b) an agent for treating the disease, wherein the agentcomprises acetylsalicylic acid or an acetylsalicylic acid derivative(e.g., with an enteric coating that allows for delivery to a subject'scolon and/or cecum).

In other embodiments, provided herein are systems comprising: a) anagent comprising acetylsalicylic acid or an acetylsalicylic acidderivative; and b) equipment that allows delivery of the agent directlyto the cecum and/or colon of a human subject.

In further embodiments, provided herein are methods for identifying TMAlyase inhibitors comprising: a) contacting a TMA-containing compound anda candidate inhibitor with a complex comprising the yeaW protein (e.g.,from E. coli) and the yeaX protein (e.g., from E. coli) under conditionsthat the complex cleaves the TMA-containing compound if not inhibited bythe candidate inhibitor; and b) determining if the complex cleaves theTMA-containing compound, wherein absence of cleavage identifies thecandidate inhibitor as a TMA lyase inhibitor.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-B show the prognostic value of plasma trimethylamine N-oxide(TMAO) levels in Chronic Kidney Disease (CKD). In a cohort of stablecardiac patients undergoing elective diagnostic coronary evaluation,subjects with underlying Stage 3+ chronic kidney disease demonstratedhigher levels of fasting plasma TMAO than those with no CKD (p<0.01,FIG. 1A). Increasing quartiles fasting plasma TMAO levels portendincreased risk for all-cause mortality at 5 years in patients with CKD(n=521, FIG. 1B).

FIG. 2 shows comparative prognostic value of plasma TMAO and Cystatin Cin patients with no Chronic Kidney Disease. Subjects with elevatedcystatin C (>0.9 mg/dL) and TMAO (>3.4 uM) had the highest 5-yearmortality risk in this non-CKD cohort (n=3,188).

FIGS. 3A-F show dietary choline/TMAO exposure contributes to progressiverenal fibrosis. Plasma TMAO (A) levels are increased after 6 weekfeeding TMAO (0.12%), or Choline (1.0%) diets vs chow (0.08% choline)fed mice. Representative Mason trichrome histology (B) quantitativemorphometry (C) and its relationship with TMAO levels (D), SMAD3activation by phosphorylation at serine 423/425 (E) and its relationshipwith TMAO levels (F) in mouse kidneys after 6 week feeding of chow(0.08% choline), TMAO (0.12%), and Choline (1.0%) diets. Scale barrepresents 100 um. **P<0.01 vs. chow fed, n>5 mice per group.

FIGS. 4A-C show that dietary choline/TMAO exposure contributes toprogressive renal injury and dysfunction. Immunoblot of KIM-1 expression(A) and its relationship with TMAO levels (B) in mouse kidneys after 6week feeding of chow (0.08% choline), TMAO (0.12%), and choline (1.0%)diets. Also shown are plasma cystatin C levels (C) after 16 week feedingof chow, TMAO (0.12%), and Choline (1.0%) diets. **P<0.01 vs. chow fed,n≥5 mice per group.

FIG. 5 show results from Example 2 and specifically shows that a cholinediet fosters a prothrombotic phenotype and that aspirin use attenuatesTMAO levels.

FIG. 6 shows results from Example 2 which shows that ASA inhibitscholine and GPC TMA lyase activity.

FIG. 7 shows results from Example 2 where ASA shows an inhibition incholine TMA lyase activity.

FIG. 8 shows results from Example 2, and specifically shows the effectof ASA (81 mg PO QD) on human subjects before and following chronicCholine supplementation. FIG. 9 shows results from Example 2, andspecifically show the effect of ASA (81 mg PO QD) on human subjectsbefore and following chronic choline supplementation.

FIG. 10 shows the nucleic acid sequence (SEQ ID NO:1) and amino acidsequence (SEQ ID NO:2) of the E. Coli yeaW gene and protein.

FIG. 11 shows the nucleic acid sequence (SEQ ID NO:3) and amino acidsequence (SEQ ID NO:4) of the E. Coli yeaX gene and protein.

FIG. 12 shows that the recombinant microbial yeaW/X complex can catalyzethe production of d9-TMA from multiple synthetic d9-trimethylamineprecursors, including yBB (gamma-butyrobetaine), L-carnitine, choline,and betaine.

FIG. 13 provides a schematic with exemplary strategies to target the gutmicrobial endocrine organ (e.g., for treating kidney disease,cardiovascular disease, or other elevated TMAO related diseases).Strategies for manipulating gut microbiota include: 1) Dietarymanipulation, 2) Prebiotics or Probiotics, 3) Fecal MicrobiotaTransplantation, 4) Antimicrobials/antibiotics, 5) Bacterial EnzymeInhibitors (e.g., TMA lyase inhibitors), or 6) Host Enzyme Inhibitors(e.g., flavin monooxygenase 3 (FMO3) inhibitors).

DEFINITIONS

As used herein, the terms “cardiovascular disease” (CVD) or“cardiovascular disorder” are terms used to classify numerous conditionsaffecting the heart, heart valves, and vasculature (e.g., veins andarteries) of the body and encompasses diseases and conditions including,but not limited to arteriosclerosis, atherosclerosis, myocardialinfarction, acute coronary syndrome, angina, congestive heart failure,aortic aneurysm, aortic dissection, iliac or femoral aneurysm, pulmonaryembolism, primary hypertension, atrial fibrillation, stroke, transientischemic attack, systolic dysfunction, diastolic dysfunction,myocarditis, atrial tachycardia, ventricular fibrillation, endocarditis,arteriopathy, vasculitis, atherosclerotic plaque, vulnerable plaque,acute coronary syndrome, acute ischemic attack, sudden cardiac death,peripheral vascular disease, coronary artery disease (CAD), peripheralartery disease (PAD), and cerebrovascular disease.

As used herein, the term “atherosclerotic cardiovascular disease” or“disorder” refers to a subset of cardiovascular disease that includeatherosclerosis as a component or precursor to the particular type ofcardiovascular disease and includes, without limitation, CAD, PAD,cerebrovascular disease. Atherosclerosis is a chronic inflammatoryresponse that occurs in the walls of arterial blood vessels. It involvesthe formation of atheromatous plaques that can lead to narrowing(“stenosis”) of the artery, and can eventually lead to partial orcomplete closure of the arterial opening and/or plaque ruptures. Thusatherosclerotic diseases or disorders include the consequences ofatheromatous plaque formation and rupture including, without limitation,stenosis or narrowing of arteries, heart failure, aneurysm formationincluding aortic aneurysm, aortic dissection, and ischemic events suchas myocardial infarction and stroke

The terms “individual,” “host,” “subject,” and “patient” are usedinterchangeably herein, and generally refer to a mammal, including, butnot limited to, primates, including simians and humans, equines (e.g.,horses), canines (e.g., dogs), felines, various domesticated livestock(e.g., ungulates, such as swine, pigs, goats, sheep, and the like), aswell as domesticated pets and animals maintained in zoos. In someembodiments, the subject is specifically a human subject.

DETAILED DESCRIPTION OF THE INVENTION

Provided herein are compositions, systems, and methods for treating adisease, such as kidney and cardiovascular disease, with an agent thatreduces the production of trimethylamine (TMA) or trimethylamine-n-oxide(TMAO) in a subject. In certain embodiments, the agent is: i)3,3-dimethyl-1-butanol (DMB) or a DMB derivative or related compound(e.g., as shown in Table 3), ii) acetylsalicylic acid or derivativethereof (e.g., with an enteric coating for delivery to the colon and/orcecum); iii) a flavin monooxygenase 3 (FMO3) inhibitor; iv) a gut TMAlyase inhibitor; v) an antibiotic or antimicrobial; vi) a probiotic orprebiotic; vii) an antiplatelet agent; or viii) a TMA and/or TMAOsequestering agent.

I. TMA and TMAO Production

The micobiota of humans has been linked to intestinal health, immunefunction, bioactivation of nutrients and vitamins, and more recently,complex disease phenotypes such as obesity and insulin resistance. Itwas recently reported that a pathway in both humans and mice linkmicrobiota metabolism of dietary choline and phosphatidylcholine tocardiovascular disease pathogenesis. Choline, atrimethylamine-containing compound and part of the head group ofphosphatidylcholine, is metabolized by gut microbiota to produce anintermediate compound known as trimethyalmine (TMA). TMA is rapidlyfurther oxidized in the liver by hepatic flavin monooxygenases to formtrimethylamine-n-oxide (TMAO), which is proatherogenic and associatedwith cardiovascular risks. These findings raise the possibility thatother dietary nutrients possessing a trimethylamine structure may alsogenerate TMAO from gut microbiota and promote acceleratedatherosclerosis.

II. DMB, Derivatives, and Related Compounds

In some embodiments, provided herein are methods for treatment and/orprevention of kidney and/or cardiovascular disease with compounds thatinhibit TMAO productions in the gut, such as 3,3-dimethyl-1-butanol(DMB), N,N-dimethylethanolamine (DMEA), N-methylethanolamine (MEA),ethanolamine (EA), trimethylsilyl ethanol, a compound from Table 3, andP,P,P-trimethyl ethanolphosphine; or other compounds represented byFormula I. Formula I is as follows:

-   wherein n is an integer, or n is 0, indicating that CH₂ is not    present;-   wherein Y is C, N, Si, P, S, Ge, Sn, Pb, P, As, Sb, or Bi;-   wherein each W is independently selected from: H, Cl, F, Br, or I    (e.g., W₃C═CH₃, CH₂Cl, CH₂Fl, CH₂Br, CH₂I, CF₃, CCl₃, CBr₃, CI₃, or    CHCl₂);-   wherein X is O or S and the corresponding bond is either present or    absent or double,-   wherein R is absent, H, an alkyl group, alkenyl group, alkynyl    group, phenyl group, amide, alkylamide, or a benzyl group;-   wherein Z is C, CH₂, CH, NH, O or S,-   wherein XR is alternatively, H, an ester, thioester, or thionester;    glycerol, or one of the following three formulas^(.)

-   wherein R′ is H, an alkyl group, alkenyl group, alkynyl group,    phenyl group, or a benzyl group; and wherein X′ is O, or S. In    certain embodiments, R is amide or alkylamide, and Z is an O, and Z    as a double bond O—a carboxylic acid). In some embodiments, the two    methyl groups extending from Y are linked by an alkyl or ether to    form a 4-6 member ring.

In some embodiments, the present invention provides methods for thetreatment and/or prevention of kidney and/or cardiovascular diseasecomprising: a) identifying a subject as having elevated TMAO and/or TMAlevels, and b) administering to the subject a composition comprisingN,N-dimethylethanolamine (DMEA), N-methylethanolamine (MEA),ethanolamine (EA), trimethylsilyl ethanol, and P,P,P-trimethylethanolphosphine, or a compound represented by Formula I (e.g.,dimethylbutanol and/or a derivative thereof), and/or a gut targetingantibiotic and/or a prebiotic (e.g. a fiber containing food that altersintestinal flora composition) and/or a probiotic (e.g., probioticcontaining food such as yogurt). In certain embodiments, the compositioncomprises dimethylbutanol or a compound shown in FIGS. 20-23 of U.S.application Ser. No. 13/915,299, which is herein incorporated byreference in its entirety. In further embodiments, the identifyingcomprises viewing results of a TMAO and/or TMA assay (e.g., on paper oron a computer screen) performed on a sample from the subject which showelevated TMAO and/or TMA levels. In certain embodiments, the identifyingcomprises viewing results of a TMA or TMAO assay performed on a sampleor exhaled breath from said subject which show elevated TMA or TMAOlevels.

In some embodiments, the composition comprises a compound of Formula I(or N,N-dimethylethanolamine (DMEA), N-methylethanolamine (MEA),ethanolamine (EA), trimethylsilyl ethanol, and P,P,P-trimethylethanolphosphine) containing food or beverage. In further embodiments,the composition comprises food or liquid containing a compound ofFormula I (or N,N-dimethylethanolamine (DMEA), N-methylethanolamine(MEA), ethanolamine (EA), trimethylsilyl ethanol, and P,P,P-trimethylethanolphosphine) selected from the group consisting of but not limitedto: olive oil, extra virgin olive oil, grape seed oil, yeast containingfood, and red wine. In other embodiments, the composition comprises acompound beneficial for reducing TMAO levels. In certain embodiments,the composition is provided in a pill or capsule (e.g., with a filler orbinder). In particular embodiments, the compound of Formula I (e.g.,dimethylbutanol) prevent TMA formation from choline or othertrimethylamine nutrients (e.g. carnitine, glycerophosphocholine,phosphocholine, phosphatodylcholine) from gut flora, or impairs cholinetransport. In additional embodiments, the compound of Formula I (orN,N-dimethylethanolamine (DMEA), N-methylethanolamine (MEA),ethanolamine (EA), trimethylsilyl ethanol, and P,P,P-trimethylethanolphosphine) induces one or more of the following when administeredto a subject: reduced trimethyl amine level, reduce total cholesterollevel, reduced LDL level, increased HDL level, and reduced triglyceridelevel. In further embodiments, the compound of Formula I reduces therisk of kidney and/or cardiovascular disease when administered to asubject.

In some embodiments, Formula I has a formula selected from the groupconsisting of:

In other embodiments, Formula I has a formula selected from the groupconsisting of:

In certain embodiments, Formula I has a formula selected from the groupconsisting of:

In some embodiments, Formula I has a formula selected from the groupconsisting of:

In further embodiments, Formula I has a formula selected from the groupconsisting of:

In some embodiments, the compounds of Formula I, or otherwise used inthe methods, systems, and compositions here, are those provided in Table3 below:

TABLE 3 Halomethyl cholines: (Fluorocholine, Chlorocholine,Bromocholine, Iodocholine) X = F, Cl, Br, I Y represents counterions:

Y = I, Br, Cl Halomethyl betaines: (Fluorodimethylglycine,Chlorodimethylglycine, Bromodimethylglycine, Iododimethylglycine)

X = F, Cl, Br, I Halomethyl betaine salts: X = F, Cl, Br, I Y representscounterions: Y = I, Br, Cl

Halomethyl betaine amides: X = F, Cl, Br, I Y represents counterions: Y= I, Br, Cl

Halomethyl betaine amides: X = F, Cl, Br, I Y = I, Br, Cl R = methyl,ethyl, propyl, amino acids, peptides

Halomethyl dimethyl amino alcohols: X = F, Cl, Br, I Y = I, Br, Cl R =methyl, ethyl, propyl (e.g. N-IodomethylN,N-dimethyl-

amine-2-hydroxy-propanol, when X = I, R = methyl) Morpholines: X = H, F,Cl, Br, I Y = Cl, Br, I (e.g. N-methyl-N-(2-hydroxyethyl) morpholine,when X = H)

Morpholines: X = H, F, Cl, Br, I Y = Cl, Br, I

III. Methods for Screening Candidate TMAO and TMA Level Lowering Agents

In some embodiments, the present invention provides drug screeningassays (e.g., to screen for TMAO and/or TMA formation inhibitor drugs).In certain embodiments, the screening methods of the present inventionutilize trimethylamine containing precursors (e.g., choline,crotonobetaine (cis and trans), gamma-butyrobetaine, carnitine,4-Trimethylammoniumbutyraldehyde, dehydrocarnitine,3-hydroxy-N6-Trimethyl-lysine, N6-Trimethyl-lysine,trimethylammoniumacetone, decarboxycarnitine, phosphocholine, betaineAldehyde, glycerophosphocholine, phosphatidylcholine, and/or betaine)incubated with intestinal microflora, or a cell-free complex ofyeaW/yeaX, capable of cleaving TMA-containing compounds to form TMA. Forexample, in some embodiments, the present invention provides methods ofscreening for compounds that inhibit the ability of the microflora fromcleaving TMA containing precursors to form TMA or using TMA to formTMAO. In some embodiments, candidate compounds are antibiotic compounds,DMB related compound, antimicrobials, candidate TMA lyase inhibitors, orcandidate FMO3 inhibitors (e.g., from a small molecule library).

In one screening method, candidate compounds are evaluated for theirability to inhibit TMA formation by microflora or a cell-free complex ofyeaW/yeaX by contacting a candidate compound with a sample containingthe microflora or a cell-free complex of yeaW/yeaX and TMA containingprecursors and then assaying for the effect of the candidate compoundson TMA formation. In some embodiments, the effect of candidate compoundson TMA formation is assayed for by detecting the level of TMA formed.

The test compounds of the present invention can be obtained, forexample, using any of the numerous approaches in combinatorial librarymethods known in the art, including biological libraries; peptoidlibraries (libraries of molecules having the functionalities ofpeptides, but with a novel, non-peptide backbone, which are resistant toenzymatic degradation but which nevertheless remain bioactive; see,e.g., Zuckennann et al., J. Med. Chem. 37: 2678-85 (1994)); spatiallyaddressable parallel solid phase or solution phase libraries; syntheticlibrary methods requiring deconvolution; the ‘one-bead one-compound’library method; and synthetic library methods using affinitychromatography selection. The biological library and peptoid libraryapproaches are generally preferred for use with peptide libraries, whilethe other four approaches are applicable to peptide, non-peptideoligomer or small molecule libraries of compounds (Lam (1997) AnticancerDrug Des. 12:145).

In certain embodiments, the test compounds are antibiotics. Any type ofantibiotic may be screened (or used to treat disease, such as kidneydisease). Examples of such antibiotics include, but are not limited to,Ampicillin; Bacampicillin; Carbenicillin Indanyl; Mezlocillin;Piperacillin; Ticarcillin; Amoxicillin-Clavulanic Acid;Ampicillin-Sulbactam; Benzylpenicillin; Cloxacillin; Dicloxacillin;Methicillin; Oxacillin; Penicillin G; Penicillin V; PiperacillinTazobactam; Ticarcillin Clavulanic Acid; Nafcillin; Cephalosporin IGeneration; Cefadroxil; Cefazolin; Cephalexin; Cephalothin; Cephapirin;Cephradine; Cefaclor; Cefamandol; Cefonicid; Cefotetan; Cefoxitin;Cefprozil; Ceftmetazole; Cefuroxime; Loracarbef; Cefdinir; Ceftibuten;Cefoperazone; Cefixime; Cefotaxime; Cefpodoxime proxetil; Ceftazidime;Ceftizoxime; Ceftriaxone; Cefepime; Azithromycin; Clarithromycin;Clindamycin; Dirithromycin; Erythromycin; Lincomycin; Troleandomycin;Cinoxacin; Ciprofloxacin; Enoxacin; Gatifloxacin; Grepafloxacin;Levofloxacin; Lomefloxacin; Moxifloxacin; Nalidixic acid; Norfloxacin;Ofloxacin; Sparfloxacin; Trovafloxacin; Oxolinic acid; Gemifloxacin;Perfloxacin; Imipenem-Cilastatin Meropenem; Aztreonam; Amikacin;Gentamicin; Kanamycin; Neomycin; Netilmicin; Streptomycin; Tobramycin;Paromomycin; Teicoplanin; Vancomycin; Demeclocycline; Doxycycline;Methacycline; Minocycline; Oxytetracycline; Tetracycline;Chlortetracycline; Mafenide; Silver Sulfadiazine; Sulfacetamide;Sulfadiazine; Sulfamethoxazole; Sulfasalazine; Sulfisoxazole;Trimethoprim-Sulfamethoxazole; Sulfamethizole; Rifabutin; Rifampin;Rifapentine; Linezolid; Streptogramins; Quinopristin Dalfopristin;Bacitracin; Chloramphenicol; Fosfomycin; Isoniazid; Methenamine;Metronidazol; Mupirocin; Nitrofurantoin; Nitrofurazone; Novobiocin;Polymyxin; Spectinomycin; Trimethoprim; Colistin; Cycloserine;Capreomycin; Ethionamide; Pyrazinamide; Para-aminosalicyclic acid; andErythromycin ethylsuccinate.

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al., Proc. Natl. Acad. Sci.U.S.A. 90:6909 (1993); Erb et al., Proc. Nad. Acad. Sci. USA 91:11422(1994); Zuckermann et al., J. Med. Chem. 37:2678 (1994); Cho et al.,Science 261:1303 (1993); Carrell et al., Angew. Chem. Int. Ed. Engl.33.2059 (1994); Carell et al., Angew. Chem. Int. Ed. Engl. 33:2061(1994); and Gallop et al., J. Med. Chem. 37:1233 (1994). Libraries ofcompounds may be presented in solution (e.g., Houghten, Biotechniques13:412-421 (1992)), or on beads (Lam, Nature 354:82-84 (1991)), chips(Fodor, Nature 364:555-556 (1993)), bacteria or spores (U.S. Patent No.5,223,409; herein incorporated by reference), plasmids (Cull et al.,Proc. Nad. Acad. Sci. USA 89:18651869 (1992)) or on phage (Scott andSmith, Science 249:386-390 (1990); Devlin Science 249:404-406 (1990);Cwirla et al., Proc. Natl. Acad. Sci. 87:6378-6382 (1990); Felici, J.Mol. Biol. 222:301 (1991)).

The ability of the test compound to inhibit TMA formation by intestinalmicroflora, or a cell-free complex of yeaW/yeaX, can be monitored bydetectably labeling the TMA portion of a TMA containing precursorcompound. Such detectable labels include, for example, radioisotopes,chromophores, fluorophores, or enzymatic labels. For example, TMAcontaining precursors can be labeled with ¹²⁵I, ³⁵S ¹⁴C or ³H, eitherdirectly or indirectly, and the radioisotope detected by direct countingof radioemmission or by scintillation counting. Alternatively, TMAcontaining precursor can be enzymatically labeled with, for example,horseradish peroxidase, alkaline phosphatase, or luciferase, and theenzymatic label detected by determination of conversion of anappropriate substrate to product. In one embodiment, the TMA containingprecursor or the test substance is anchored onto a solid phase. The TMAcontaining precursor anchored on the solid phase can be detected at theend of the reaction.

In certain embodiments, cell free assays can be conducted in a liquidphase using a complex of yeaW/yeaX. In such an assay, the reactionproducts are separated from unreacted components, by any of a number ofstandard techniques, including, but not limited to: differentialcentrifugation (see, for example, Rivas and Minton, Trends Biochem Sci18:284-7 (1993)); chromatography (gel filtration chromatography,ion-exchange chromatography); electrophoresis (see, e.g., Ausubel etal., eds. Current Protocols in Molecular Biology 1999, J. Wiley: NewYork.); and immunoprecipitation (see, for example, Ausubel et al., eds.Current Protocols in Molecular Biology 1999, J. Wiley: New York). Suchresins and chromatographic techniques are known to one skilled in theart (See e.g., Heegaard J. Mol. Recognit 11:141-8 (1998); Hageand TweedJ. Chromatogr. Biomed. Sci. Appl 1 699:499-525 (1997)).

This invention further pertains to agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model to determine the efficacy, toxicity, sideeffects, or mechanism of action, of treatment with such an agent.Furthermore, novel agents identified by the above-described screeningassays can be used for treatments as described herein.

EXAMPLES

The following examples are for purposes of illustration only and are notintended to limit the scope of the claims.

Example 1 The Gut Microbiota-Dependent TMAO Pathway Contributes toDevelopment of Renal Insufficiency and Mortality Risk in Chronic KidneyDisease

This example describes an examination of the contribution of TMAO to thedevelopment of renal insufficiency and mortality risk in chronic kidneydisease (CKD).

Methods

Human studies. The human population studied is a single-center,prospective cohort approved by the Cleveland Clinic Institutional ReviewBoard. Adult subjects (ages 18 years and above) were included whounderwent elective diagnostic coronary angiography for cardiacevaluation from 2001-2007 as previously described (9). Subjects withknown acute coronary syndromes or revascularization procedures within 30days of enrollment, or history of congenital heart disease, wereexcluded. After informed consent, fasting plasma blood samples werecollected using ethylenediaminetetraacetic acid tubes prior to any drugadministration via the arterial sheath, and immediately processed andfrozen in −80° C. until analysis. Estimated glomerular filtration rate(eGFR) was calculated according to the CKD-EPI creatinine and cystatin Cformula (17), with CKD defined as eGFR >60 ml/min/1.73 m2 (CKD stage 3or beyond). Ascertainment of all-cause mortality at 5-years wasperformed by prospective telephone contact and chart review plusinterrogation of the Social Security Death Index (up to 2011).

Plasma analysis. Trimethylamine-N-oxide (TMAO) levels were determined bystable isotope dilution high-performance liquid chromatography withonline electrospray ionization tandem mass spectrometry (LC/MS/MS) on anAB SCIEX 5500 triple quadrupole mass spectrometer (AB SCIEX, FraminghamMA) using d9-(trimethyl)-labeled internal standards as previouslydescribed (10, 18). High-sensitivity C-reactive protein (hsCRP), fastinglipid panel, cystatin C, and serum creatinine were measured using theArchitect ci8200 platform (Abbott Laboratories, Abbott Park, Ill.).

Animal study. To directly test for a potential contribution of dietarycholine or TMAO to promotion of renal dysfunction, C57BL6J mice were fedwith the following diets for 6 weeks: i) a chemically defined dietcomparable in composition to standard chow diet (Teklad 2018, HarlandLaboratories) that contains 0.08% (gm/gm) total choline; ii) the samediet supplemented with choline (1% total); and iii) the same dietsupplemented with TMAO (0.12%). A separate study included C57BL6J micewith ApoE−/− background were fed with the same diet groups for comparingtheir cystatin C levels at 14 weeks of follow-up using a commerciallyavailable mouse enzyme-linked immunosorbent assay (R&D systems,Minneapolis Minn.). This study has been approved by the Cleveland ClinicInstitutional Animal Care and Use Committee.

Quantitative Histologic Techniques. Mason's trichome staining wasperformed on deparafinized 5 μm serial kidney sections. The kidneysections were mounted under a Leica DM 2500 microscope and digitizedwith a Qlmaging MicroPublisher 5.0 RTV camera for wide field microscopy.Quantitative morphometric analysis was performed on cortical fields (atleast 8 from each animal) lacking major blood vessels and the collagenvolume was determined using automated (for batch analysis) andcustomized algorithms/scripts (ImageIQ Inc., Cleveland, Ohio) writtenfor Image Pro Plus 7.0. Briefly, a set of representative images arechosen that demonstrated a wide range of staining intensities andprevalence. In an automated script, these “training” images were loadedone after another prompting the user to delineate “blue” pixelsrepresenting positive collagen staining using an interactive colorpicking tool. An iterative color profile or classifier was generated andsubsequently applied to all images in a given directory using a fullyautomated algorithm. Positive pixels, as defined by the color profile,were segmented and summed to provide positive staining area. Totaltissue area was determined by extracting the “saturation” channel,applying a lo-pass filter, and thresholding the result. Any area withinthe general tissue boundary that was empty (i.e. white) was removed byconverting the original image to grayscale and applying a fixedthreshold for non background pixels on adequately white-balanced images.Finally, total tissue area and total stained area were exported toExcel. For post-processing verification, segmented regions weresuperimposed onto the original image (green outlines) and saved for eachimage analyzed.

Preparation of tissue homogenates and immunoblotting. Equal amounts ofprotein were prepared using standard biochemical methods and subjectedto SDS-PAGE and electrotransfer of proteins from gels to Immobilon-Pmembranes (Millipore). Membranes were incubated with the followingantibodies: SMAD3 and phospho-SMAD-3(Ser423/425) (Cell SignalingTechnology); tubulin (Santa Cruz Biotechnology); KIM-1 (NovusBiologicals). Detection for all immunoblots was performed with the SNAPi.d.™ Protein Detection System (Millipore) and Super SignalChemiluminescent Substrate Products (Pierce), and band intensity wasanalyzed by densitometry (ImageQuant™, GE Healthcare).

Statistical analyses. Continuous variables were summarized asmean±standard deviation if normally distributed and median(interquartile ranges [IQR]) if non-normally distributed. The student'st-test or Wilcoxon-Rank sum test for continuous variables and chi-squaretest for categorical variables were used to examine the differencebetween groups. Spearman's correlation was used to examine theassociations between TMAO and other laboratory measurements.Kaplan-Meier survival plots and Cox proportional hazards analysis wereused to determine Hazard ratio (HR) and 95% confidence intervals (95%CI)for all-cause mortality stratified according to TMAO in quartiles.Adjustments were made for individual traditional risk factors includingage, sex, systolic blood pressure, low-density lipoprotein cholesterol(LDLc), high-density lipoprotein cholesterol (HDLc), smoking, diabetesmellitus, log-transformed hsCRP, and logtransformed eGFR to predictall-cause mortality risks. Net reclassification and area under ReceiverOperator Characteristic curve were calculated according to mortalityrisk estimated using Cox models adjusted for above mentioned traditionalrisk factors with versus without TMAO as previously described19. Allanalyses performed used R 2.15.1 (Vienna, Austria). P<0.05 wasconsidered statistically significant.

Results

Elevated TMAO in Patients with Renal Insufficiency Portend PoorerSurvival. Baseline clinical and laboratory characteristics of the cohortare reported in Table 1.

TABLE 1 Baseline Characteristics eGFR ≥0.60 eGFR <60 Characteristic (n =3,166) (n = 521) p-value Age (years) 62 ± 11 70 ± 10 <0.001 Male (%) 6648 <0.001 Diabetes (%) 27 53 <0.001 Hypertension (%) 69 88 <0.001Smoking (%) 66 61 0.047 History of MI (%) 40 53 <0.001 History of stroke(%)  5 13 <0.001 History of CABG (%) 28 42 <0.001 History of PCI (%) 3130 0.728 LDL (mg/dL) 97 (79-118) 93 (72-114) <0.001 HDL (mg/dL) 34(28-41) 32 (26-40) <0.001 hsCRP (mg/L) 2.2 (0.9-5.0) 4.1 (1.8-9.6)<0.001 eGFR (ml/min/1.73 m²) 89 (78-101) 49 (38-55) <0.001 Cystatin C(mg/L) 0.9 (0.8-1) 1.5 (1.3-1.8) <0.001 ACE inhibitor/ARB (%) 48 66<0.001 Statins (%) 61 59 0.437 Beta blockers (%) 63 68 0.04 Aspirin (%)75 67 <0.001 TMAO (μM) 3.4 (2.3-5.3) 7.9 (5.2-12.4) <0.001

A total of 3,687 subjects were included in this analysis, among which521 subjects fulfilled criteria for CKD and 3,166 subjects for non-CKD.Compared to non-CKD subjects, TMAO levels were elevated in patients withCKD (median TMAO: 7.9 [IQR 5.2-12.4]uM versus 3.4 [IQR 2.3-5.3]uM,p<0.001; FIG. 1A). Overall in the CKD cohort, TMAO modestly correlatedwith eGFR (r=−0.48, p<0.001) and cystatin C (r=0.46, p<0.001), but onlyweakly correlated with hsCRP (r=0.04, p=0.332).

In the CKD cohort, higher TMAO levels (quartiles 4 versus 1) wereassociated with a 2.8-fold increase in risk for all-cause mortality at 5years (unadjusted HR 2.76, 95%CI 1.74-4.37, p<0.001). After adjustingfor traditional CVD risk factors, log-transformed hsCRP, andlog-transformed eGFR, higher TMAO levels still were associated with a1.9-fold poorer 5-year survival (adjusted HR 1.93, 95%CI 1.13-3.29,p<0.05; Table 2, Kaplan-Meier curve shown in FIG. 1B).

TABLE 2 Cox Proportional Hazards Analysis of Plasma TMAO LevelsStatified at quartile Levels in Predicting Risk of All-cause Mortalityat 5 Years Stratified by CKD and Non-CKD Cohorts TMAO (range) Quartile 1Quartile 2 Quartile 3 Quartile 4 CKD Cohort (n = 521) Range (μM) <5.25.2-7.9 7.9-12.4 ≥12.4 Events 26/129 42/131 43/130 63/131 (20.2%)(32.1%) (33.1%) (48.1%) Unadjusted HR 1 1.70 1.75 2.76  (1.04-2.79)*(1.07-2.87)*  (1.74-4.37)** Adjusted HR 1 1.42 1.51 1.93 (0.85-2.35)(0.90-2.51)  (1.13-3.29)*  Non-CKD Cohort (n = 3166) Rarge (mM) <2.32.3-3.4 3.4-5.3 ≥5.3 Events 48/787 59/793 81/791 104/795 (6.1%) (7.4%)(10.2%) (13.1%) Unadjusted HR 1 1.22 1.7 2.21 (0.83-1.79) (1.19-2.43)**0 .57-3.12)** Adjusted HR 1 1.08 1.23 1.47 (0.74-1.56) (0.84-1.78) (1.02-2.12)* 

When stratified according to median levels (7.9 uM), higher TMAOconferred a 1.7-fold increase in risk for all-cause mortality (HR 1.70,95%CI 1.25-2.30, p<0.001), and remained significant after adjusting fortraditional risk factors and log-transformed hsCRP (adjusted HR 1.72,95%CI 1.16-2.34, p<0.001), as well as with addition of cystatin C to themodel (adjusted HR 1.45, 95%CI 1.05-2.02, p<0.05). Using median cohortcut-offs with low cystatin C (<1.4 mg/dL) and low TMAO (<7.9 uM) asreference, those with concomitant high cystatin C and high TMAO had a3-fold increase in mortality risk (HR 3.01, 95%CI 1.97-4.59, p<0.001).These findings are consistent with the notion that elevated TMAO isassociated with poor prognosis in patients with established CKD.

Increased TMAO Levels in Non-CKD Patients with Elevated Cystatin C.Within the non-CKD cohort (n=3,166), the prognostic value of elevatedTMAO (quartile 4 vs 1) remained predictive of 5-year mortality risk (HR2.21, 95%CI 1.57-3.12, p<0.001), as well as after adjusting fortraditional risk factors, log-transformed hsCRP, and logtransformed eGFR(adjusted HR 1.47, 95%CI 1.02-2.12, p<0.05, Table 2B). These findingswere similar when restricted to subjects with preserved eGFR (≥60ml/min/1.73 m2) plus normal cystatin C (<1.4 mg/dL, n=3,151). ElevatedTMAO levels is associated with higher 5-year mortality risk in bothnormal and elevated cystatin C levels (FIG. 2). Using median cohortcut-offs with low cystatin C (<0.9 mg/dL) and low TMAO (<3.4 uM) asreference, those with concomitant high cystatin C and high TMAO had a3.7-fold increase in mortality risk (HR 3.67, 95%CI 2.57-5.23, p<0.001).

Dietary Choline and TMAO Promotes Renal Fibrosis and Dysfunction inAnimal Models.

To directly test the hypotheses that either dietary TMAO itself, ordietary nutrients that contribute to gut microbiota dependent productionof TMAO, can impact development and progression of CKD, animal modelstudies were performed. Conventionally housed 8-week old male mice(C57BL/6J background) were fed ad libitum a chemically defined dietcomparable to normal chow (0.08 gm % choline), or the same dietsupplemented with either choline (1.0% final) or TMAO (0.12%), asdescribed under Methods. After 6 weeks, significant (p<0.01) increaseswere observed in TMAO levels in both the TMAO supplemented andcholine-supplemented groups of mice (FIG. 3A), with TMAO levels observedwithin the range of values detected among CKD subjects studied (97.5percentile 77.6 uM, 99 percentile 96.3 uM). Importantly, elevated TMAOlevels were associated with corresponding increases in parenchymalfibrosis and collagen deposition (FIG. 3B-C) and phosphorylation ofSmad3, an important regulator of the pro-fibrotic TGF-β/Smad3 signalingpathway during fibrotic kidney disease20 (FIG. 3E). Furthermore,TMAO-fed and choline-fed mice experienced increased kidney injurymarker-1 (KIM-1, FIG. 4A). Extending the TMAO/choline feeding to 16weeks was associated with increased serum cystatin C levels compared tochow-fed mice (FIG. 4C). Upon further examination, strikingdose-dependent relationships were noted between plasma TMAO levels andmonitored indices of renal histopathological (FIG. 3D, 3F) andfunctional impairment (FIG. 4B).

Discussion

There are several key findings in this Example. First, in subjects withCKD, it was observed that TMAO levels are not only elevated compared tonon-CKD subjects, but importantly, portend poorer overall survival.Second, it was observed that within the non-CKD cohort, higher levels ofTMAO portend poorer survival both within the cohort of low levels aswell as high levels of cystatin C (stratified at median levels).Interestingly, the prognostic value for the highest TMAO quartile inpredicting future mortality risk in this cohort remained robust evenafter adjustment for traditional risk factors. Third, extending toanimal models studies, dietary exposure of either choline or TMAO leadto the development of renal parenchymal fibrosis and early measures ofdysfunction (elevated cystatin C). These studies thus suggest both acausal relationship and clinical relevance of dietary choline-induced,gut microbiota-mediated TMAO formation in CKD development andprogression.

Trimethylamine N-oxide is a low molecular weight compound that is easilyfiltered by the kidney, and effectively removed by hemodialysis (13).Considered a nitrogenous waste product whose levels rise with diminishedrenal function, elevated TMAO levels have been reported in small cohorts(n<20) of subjects with either end-stage renal disease, or CKD, wherelevels were shown to correlate with both serum urea and creatinine (15).Detailed animal and human experiments on the renal clearance ofmethylamines such as TMA and TMAO have been performed, confirming thekidneys as the primary elimination route (21). Interestingly, theurinary clearances of both TMA and TMAO are higher than the glomerularfiltration rate, and TMAO clearance also decreases with increasing dose,which suggests that saturable renal tubular secretion occurs (22). Themajority of a dose of TMA is also excreted in the urine, with varyingproportions in the forms of TMA and TMAO being dependent on the doselevel (23). Urinary TMAO levels are reported to rise with episodes ofkidney graft dysfunction in renal transplant recipients, suggesting anintrinsic accumulation of TMAO (presumably as an osmolyte like urea)that is released during damage of the renal medullary cells (24-27). Themechanistic link between elevated TMAO and adverse prognosis in CKD andeven in the setting of subclinical renal insufficiency (elevatedcystatin C) in non-CKD patients observed in this Example is thereforeconsistent with the heightened risk of developing CKD in the CKDpopulation.

These results from animal studies showed for the first time a directmechanistic link between dietary choline, or dietary TMAO, andprogressive renal fibrosis and dysfunction. Indeed, both brief exposure(6 weeks) to either a high choline diet or a diet supplemented directlywith TMAO both led to increased levels of the early kidney injury markerKIM-1, and enhanced phosphorylation of Smad3, an important regulator ofrenal fibrosis (28). A more prolonged exposure to either the highcholine diet or the TMAO supplemented diet both led to increased plasmalevels of cystatin C, a sensitive indicator of renal functionalimpairment. Interestingly, a recent untargeted metabolomic study fromthe Framingham Heart Study identified elevated choline and TMAO levelswere associated with an increased future risk of developing CKD (29).The animal model findings of this Example therefore provide a potentialmechanistic rationale for the Framingham observational data, andcollectively, further link elevated TMAO levels with increasedsusceptibility of CKD.

The prospects that exposure to specific dietary nutrients via gutmicrobiota may impact susceptibility to the development and progressionof both CKD and CVD has important potential public health implications.Randomized nutritional intervention studies in CKD patients to date havenot explored a potential role for choline, phosphatidylcholine,L-carnitine or TMAO (which can be abundant in certain types of fish) indisease progression. Similarly, epidemiological studies are ratherlimited on the topic of diet and CKD risks, even though a recommendedrenal diet is typically low in protein intake.

Dietary management of CKD patients represents a challenge, and much lessis known about nutritional factors that might predispose to enhancedrisk for development of CKD or its progression. Interestingly, in asub-study (n=3,296) amongst women who had urine microalbumin levelsavailable from the Nurses Health Study, two or more servings of red meat(primary source of L-carnitine) per week were directly associated withenhanced risk for development of microalbuminuria (OR: 1.51; 95% CI:1.01 to 2.26) (30. Based upon the present Example, a diet monitored byfollowing TMAO levels and designed to limit TMAO precursors (low in redmeat, meats, liver, egg yolk, and high fat dairy products) and TMAOitself (certain fish) would be an attractive diet to limit the rate ofCKD progression.

Collectively, the present data indicate a dietary-induced, intestinalmicrobiota-dependent mechanism contributes to both progressive renalfibrosis and dysfunction, and mortality risks, among subjects with CKD.They also build upon the recent body of evidence demonstrating amechanistic link between gut microbiota-associated metabolicdysregulation and cardiovascular risk in humans (8-11). The discovery ofthe metaorganismal pathway involved in TMAO generation thus affords aunique opportunity to systematically investigate the potentialcontributions of discrete participants in the overall diet→microbe→hostenzyme pathways for TMAO formation and development and progression ofcardio-renal dysfunction, thereby offering insights into modulation ofsuch pathways. It is interesting that in both animal models and patientswith established CKD, pre- and probiotic intervention studies have beenperformed, with reports of changes in gut microbiota composition andactivity. For example, lactobacillus acidophilus or bifidobacterium havebeen reported to reduce inflammatory signaling associated with themicrobiota-derived metabolites that accumulate in CKD (31-34), inaddition to modestly improving renal function (35, 36). Similarly,prebiotic compound use to interrupt pathways that lead to gut microbiotaderived uremic toxins such as indoxyl sulfate and p-cresyl sulfate hasshown some efficacy in both human and animal trials of CKD (5, 37).

Example 2 ACE (Acetylsalicylic Acid) Reduction of TMAO Production InVivo

This Example investigated the question of whether aspirin can aspirin(ASA) mitigate the prothrombotic phenotype associated with high cholineor TMAO related diet. And if so, is there evidence that aspirin therapyitself impacts TMAO production in vivo, and could this be a mechanismthrough which aspirin promotes some of its cardiovascular benefit.

In the first part of this Example, the impact of aspirin (ASA) therapyon the prothrombotic phenotype provoked by chronic cholinesupplementation in the diet was examined. This diet elevates TMAO, andprovokes enhanced platelet responsiveness and thrombosis. C57BL/6Jfemales mice were placed on a chemically defined diet comparable to thenormal mouse feed (chow) vs. an identical diet supplemented with 0.5 gm% choline (choline). Each of these two groups of mice were then split,and half placed on aspirin therapy at a dose comparable to that used inhumans (ASA 4 mg/Kg BW in drinking water). After 2 weeks, plasma wascollected for TMAO levels by established stable isotope dilutionLC/MS/MS analyses. In parallel, in vivo thrombosis potential wasmeasured using the well established carotid artery FeCl exposure modelwith vital microscopy. In this model, the internal carotid artery isexposed by cutdown, and visualized by computer imaging software usingvital microscopy. Platelets are removed and fluorescently labeled, andthen infused in the contralateral jugular vein. Following FeCl exposure,one can monitor clot formation directly over time by visualizing thecarotid artery at the site of FeCl application. One can also monitorblood flow down stream from the clot formation. Thus, one can bothquantify clot formation, and time to blood flow cessation downstreamfrom the clot. Either of these represent quantitative indices ofthrombosis potential in vivo.

FIG. 5 shows the results of this work. Along the bottom in FIG. 5, thelevels of TMAO monitored in plasma are displayed. Note first thatcholine diet induced a significant increase in TMAO (8.3 vs 43.31 uM;p<0.05 for chow vs choline without aspirin(ASA)). A similar increase inTMAO is noted in mice on ASA concurrently with choline diet (4.5 vs19.7; p<0.05 for chow vs choline groups on ASA). Also note that in bothdiets (chow and choline) addition of ASA resulted in significantreduction in TMAO level (approx. 2-fold; p<0.05 for comparison in eachdiet). Thus, these studies show that ASA therapy lowers TMAO levels. Inthe in vivo thrombosis studies, it is noted that choline diet alonereduces the time to blood flow cessation (prothrombotic; p<0.0001).However, addition of ASA to the choline diet attenuates the extent ofprothrombotic phenotype observed. For example, the choline diet+ASAgroup shows no difference in time to blood flow cessation relative tothe control (chow and no ASA) group. Of note, the addition of cholineinto the diet still impacts time to blood flow cessationand hencepromotes a prothrombotic effect still in ASA treated groups, even thoughthe ASA promotes an improvement (comparison of chow+ASA vs choline+ASAreveals significant prothrombotic effect of choline diet still with ASAtherapy).

Collectively, this work indicates: 1) in the setting of elevated TMAO,where a prothrombotic effect is generated, addition of aspirin maymitigate the adverse effect. 2) ASA therapy can be used to lower TMAO.This suggests ASA can be used, for example, as a therapy to inhibitmicrobial TMA lyases, the rate limiting step in TMAO production in vivo.3) Elevated TMAO levels may serve as an indication for antiplateletrelated interventions, such as aspirin, and other antiplatelet drugs(e.g., clopidegrel, prasugrel); abciximab (Reopro®); Aggrenox®(dipyridamole/ASA); anagrelide (Agrylin®); cilostazol (Pletal®);clopidogrel (Plavix®); dipyridamole (Persantine®); eptifabatide(Integrilin®); prasugrel—EFFIENT™; ticagrelor (BRILINTA™); ticlopidine(Ticlid®); tirofiban (Aggrastat®); and vorapaxar (ZONTIVITY™).

Based on the findings that ASA therapy in vivo reduced TMAO, andknowledge that TMA lyases represent the major determinant of TMA andhence, TMAO levels, it was tested whether ASA directly could impactmicrobial TMA lyase activity. Proteus mirabilis (PM) is a microbe withabundant choline TMA lyase activity that studies suggest serves as anexcellent source of microbial TMA lyase activity similar to thatobserved in vivo (within cecum and colon of rodents and humans, andfeces of rodents and humans). Using PM lysate (PM that is lysed in aFrench press to break open the cell wall and release enzyme contents) asthe enzyme source, the TMA lyase activity (TMA producing activity) wasexamined in incubations with either d9(trimethyl)choline as substrate,or d9(trimethyl) glycerophsopphocholine (GPC) as substrate. In bothcases, it was noted that addition of a physiological range of ASAinhibited TMA production from the substrates. These data directlydemonstrate aspirin can inhibit microbial TMA lyase activity and likelypromotes TMAO reduction in vivo in part via direct inhibition in themicrobial TMA lyases in vivo. The results of this work are shown in FIG.6.

A further examination of the impact of ASA on TMA lyase activity by PMlysate was undertaken. Three mg of PM lysate was incubated with 25 uMcholine and 25 uM choline(d9) in 2 ml PBS at 37 C for 20 hours. In FIG.7, the dotted lines at 81 mg and 320 mg ASA represent reported steadystate plasma levels of aspirin reported at those doses in humansubjects. Thus, at a dose of one aspirin a day (320 mg) plasma levels ofASA anticipated result in approximately 40% TMA lyase inhibition. Inlight of this, in some embodiments, enteric coated aspirin or aspirinderivatives that have delayed absorption may be employed to treatdisease since they would dissolve in the intestines past the stomach,such that the level of ASA or derivative delivered to the gut lumen willbe higher. There exist even longer delayed release formulations ofmedications that could also be used. For example, an aspirin analog, 5amino salicylic acid, could be used for treatment of diseases withexcess TMAO production in the gut. Not only are significantly higherlevels of ASA achievable within the distal gut lumen by this approach,but in addition to delayed release formulations, prodrug formulationsreleased by gut microbe action in the ilium and colon are available andcould be employed (see, e.g., Williams et al., Therap Adv Gastroenterol.July 2011; 4(4): 237-248; herein incorporated by reference, whichoutlines some of these formulations and applications that have beenapplied to 5-ASA, and thus can be applied to ASA as well).

In further works, human volunteer subject were enrolled in the followingstudy. Individuals were examined at baseline for plasma TMAO and blooddrawn for platelet aggregometry studies with submaximal ADP stimulation.Subjects were examined both naive to aspirin, and then at different timeafter taking chronic aspirin (81 mg daily) for at least a month. Asshown in FIG. 8, at baseline, looking just at those naive to aspirin,addition of choline supplementation in the diet (equivalent to thecholine content of 2 hard boiled eggs daily—see below) showed anaccompanying rise in plasma TMAO levels at 1 and 2 months point, andcorresponding increases in platelet aggregometrry. After washout periodof at least 4 months with no choline supplement, subjects were placed onASA daily (81 mg) and after a month of ASA, the baseline TMAO level andplatelet aggregmetry measure monitored. Then, choline supplementationdaily was again resumed. The impact of the choline diet on rise in TMAOwas blunted. And the corresponding rise in platelet aggregation too wasblunted. All subjects presented in FIG. 8 were omnivores.

These studies show ASA therapy helps to lower TMAO levels, and reducesprothrombotic effect associated with high choline diet (high TMAO). Theyunderscore that TMAO elevation also may be an indication for ASAprophyllaxis therapy to prevent platelet hyperreactivity and clot riskor to treat other elevated gut TMAO diseases.

FIG. 9 shows the same type of data as FIG. 8, except vegans wereexamined before vs after starting choline supplementation. Cholinesupplement was used in subjects (choline bitartrate, 2×500mg/day=choline 2×205.7 mg/day), for 2 month period. Samples werecollected at baseline and monthly blood were draw (1,2 months). Plasmalevels of TMAO were measured and platelet aggregation in response to 5uM ADP were measured. It was observed that the vegan diet does notprotect the subject completely from elevation in TMAO from choline diet,nor a significant increase in platelet hyperresponsiveness from cholinediet.

Example 3 Identification of yeaW and yeaX as TMA Lyases

As part of this example, microbial enzymes of unknown function weresearched for that were clustered with those known to synthesize or useeither malate or succinate (potential products formed following carntineutilization) and a presumed betaine-carnitine-choline transporter. Onepotential candidate was gene pair previously called yeaW/X in E. ColiDH10B (yeaW (dioxygenase), GeneID: 6060925 and yeaX (oxidoreductase),GeneID: 6060982). Using a modified pET20 plasmid, E. coli BL21pLysS wastransformed with each allele and subsequently individually purifiedrecombinant yeaW and yeaX from bacterial lysates using the 8xH is metalaffinity chromatography. When the purified proteins are combined, theyeaW/X complex demonstrated carnitine TMA lyase activity (monitored byd9-TMA production from d9-carnitine). Interestingly, furthercharacterization of the recombinant microbial yeaW/X complex revealedsubstrate promiscuity, catalyzing production of d9-TMA from multiplesynthetic d9-trimethylamine precursors (yBB, L-carnitine, choline, andbetaine; FIG. 12).

The cloning, expression, isolation and characterization of yeaW/yeaX wasas follows. A gene cluster consistent with the features sought wasidentified including the genes yeaU (dehydrogenase), GeneID: 6060908;yeaV (putative BCCT transporter), GeneID: 6060973; yeaW (dioxygenase),GeneID: 6060925 and yeaX (oxidoreductase), GeneID: 6060982. The genesfor yeaW and yeaX are contiguous and were PCR'd from genomic E. ColiDH10B DNA, GeneBank: CP000948.1, nucleotides 1973260-1975405 using thefollowing forward and reverse primers, respectively, (gene-specificsequences are underlined)

(SEQ ID NO: 5) 5′AGGAGATACCATGAGCAATCTGAGCCCTGACTTTGTACTAC, and (SEQ ID NO : 6) 5′AGGAGATATACCATGTCAGACTATCAAATGTTTGAAGTACAGGTG.PCR reactions were run in 50 ul aliquots using the following temperatureprogram: 2 min at 95° C.; 1×(20 sec at 95° C. 30 sec at 57° C., 80 secat 68° C.); 30×(20 sec at 95° C., 30 sec at 62° C., 80 sec at 68° C.); 3min at 68° C. and 4° C. hold. PCR reactions were fractionated on a 1%agarose gel and the PCR fragment gel-purified, quantified and used forcloning into a modified pET20 vector at the Nco I/HindIII sites usingInFusion (Clontech). After DNA sequence verification of clones, pilotexpression studies confirmed that the correct size protein(s) were beingproduced and that the lysate, but not lysate from induced native BL21cells, had carnitine TMA-lyase activity (produced d9-TMA fromd9-carnitine). The individual yeaW and yeaX genes were then PCR'd fromthis expression clone using gene-specific primers and cloned in-framebehind an 8×-His tag in a modified pET20 vector. Upon sequenceverification, the individual epitope-tagged yeaW and yeaX alleles weretransformed into E. Coli BL21 DE3 pLysS cells (Invitrogen) andindividual colonies of each were expanded for growth, induction,harvesting and extract preparation, as previously described (Gogonea etal., 2010, Biochemistry 49, 7323-7343). Cells expressing 8×-His-taggedyeaW or yeaX were grown to an OD600 of 0.5, induced at room temperaturewith 0.4 mM IPTG for 18 h, and then harvested and protein extractsprepared using a microfluidizer, as previously described (Gogonea etal., 2010). Recombinant proteins were purified using IMAC Ni-chargedresin, as previously described (Gogonea et al., 2010). Enzymaticactivity was determined by incubation of the purified yeaW and eaX (30ug each) with 100 uM deuterium labeled trimethylamine containingcompounds in the presence of 200 uM NADH in 1 ml PBS at 37oC in gastight vials. The product, d9-TMA, was determined by LC/MS/MS asdescribed above.

REFERENCES

-   1. Mafra et al., Role of altered intestinal microbiota in systemic    inflammation and cardiovascular disease in chronic kidney disease.    Future Microbiol 2014; 9:399-410.-   2. Vaziri et al., Chronic kidney disease alters intestinal microbial    flora. Kidney Int 2013;83(2):308-15.-   3. Stenvinkel and Alvestrand, Inflammation in end-stage renal    disease: sources, consequences, and therapy. Semin Dial 2002;    15(5):329-37.-   4. Anders et al., The intestinal microbiota, a leaky gut, and    abnormal immunity in kidney disease. Kidney Int 2013;83(6):1010-6.-   5. Lee et al., Effects of AST-120 on blood concentrations of    protein-bound uremic toxins and biomarkers of cardiovascular risk in    chronic dialysis patients. Blood Purif 2014;37(1):76-83.-   6. Lekawanvijit et al., Cardiorenal syndrome: the emerging role of    protein-bound uremic toxins. Circ Res 2012;111(11):1470-83.-   7. Ramezani and Raj. The gut microbiome, kidney disease, and    targeted interventions. J Am Soc Nephrol 2014;25(4):657-70.-   8. Koeth et al., Intestinal microbiota metabolism of L-carnitine, a    nutrient in red meat, promotes atherosclerosis. Nat Med 2013;    19(5):576-85.-   9. Tang et al., Intestinal microbial metabolism of    phosphatidylcholine and cardiovascular risk. N Engl J Med 2013;    368(17):1575-84.-   10. Wang et al., Gut flora metabolism of phosphatidylcholine    promotes cardiovascular disease. Nature 2011; 472(7341):57-63.-   11. Wang et al., Prognostic value of choline and betaine depends on    intestinal microbiotagenerated metabolite trimethylamine-N-oxide.    Eur Heart J 2014; 35(14):904-10.-   12. Bennett et al. Trimethylamine-Noxide, a metabolite associated    with atherosclerosis, exhibits complex genetic and dietary    regulation. Cell Metab 2013; 17(1):49-60.-   13. Bain et al., Accumulation of trimethylamine and    trimethylamine-N-oxide in end-stage renal disease patients    undergoing haemodialysis. Nephrol Dial Transplant 2006;    21(5):1300-4.-   14. Bain et al., Oral L-carnitine: metabolite formation and    hemodialysis. Curr Drug Metab 2006; 7(7):811-6.-   15. Bell et al., Nuclear magnetic resonance studies of blood plasma    and urine from subjects with chronic renal failure: identification    of trimethylamine-N-oxide. Biochim Biophys Acta 1991; 1096(2):101-7.-   16. Robert et al., A pair analysis of the delayed graft function in    kidney recipient: the critical role of the donor. Journal of    critical care 2010; 25(4):582-90.-   17. Inker et al.,. Estimating glomerular filtration rate from serum    creatinine and cystatin C. N Engl J Med 2012; 367(1):20-9.-   18. Wang et al., Measurement of trimethylamine-N-oxide by stable    isotope dilution liquid chromatography tandem mass spectrometry.    Anal Biochem 2014; 455:35-40.-   19. Pencina et al., Evaluating the added predictive ability of a new    marker: from area under the ROC curve to reclassification and    beyond. Stat Med 2008; 27(2):157-72; discussion 207-12.-   20. Qu et al., Regulation of renal fibrosis by Smad3 thr388    phosphorylation. Am J Pathol 2014; 184(4):944-52.-   21. Al-Waiz et al., The metabolism of 14C-labelled trimethylamine    and its N-oxide in man. Xenobiotica 1987; 17(5):551-8.-   22. Smith et al., Metabolism and excretion of methylamines in rats.    Toxicol Appl Pharmacol 1994; 125(2):296-308.-   23. Zeisel et al., Conversion of dietary choline to trimethylamine    and dimethylamine in rats: dose-response relationship. J Nutr 1989;    119(5):800-4.-   24. Hauet et al., Proton NMR spectroscopy as a novel approach to the    monitoring of citrate and trimethylamine-N-oxide excretion after    kidney preservation. Transplant Proc 1997; 29(5):2323-5.-   25. Le Moyec et al., Proton nuclear magnetic resonance spectroscopy    of urine and plasma in renal transplantation follow-up. Nephron    1993; 65(3):433-9.-   26. Serkova et al., H-NMR-based metabolic signatures of mild and    severe ischemia/reperfusion injury in rat kidney transplants. Kidney    Int 2005; 67(3):1142-51.-   27. Foxall et al., NMR spectroscopy as a novel approach to the    monitoring of renal transplant function. Kidney Int. 1993;    43(1):234-45.-   28. Runyan et al., Smad3 and PKCdelta mediate TGF-betal-induced    collagen I expression in human mesangial cells. Am J Physiol Renal    Physiol 2003; 285(3):F413-22.-   29. Rhee et al., A combined epidemiologic and metabolomic approach    improves CKD prediction. J Am Soc Nephro! 2013; 24(8):1330-8.-   30. Lin et al., Associations of diet with albuminuria and kidney    function decline. Clin J Am Soc Nephrol 2010; 5(5):836-43.-   31. Hegazy and El-Bedewy, Effect of probiotics on pro-inflammatory    cytokines and NF-kappaB activation in ulcerative colitis. World J    Gastroenterol 2010; 16(33):4145-51.-   32. Seth et al., Probiotics ameliorate the hydrogen peroxideinduced    epithelial barrier disruption by a PKC- and MAP kinase-dependent    mechanism. Am J Physiol Gastrointest Liver Physiol 2008;    294(4):G1060-9.-   33. Simenhoff et al., Biomodulation of the toxic and nutritional    effects of small bowel bacterial overgrowth in end-stage kidney    disease using freeze-dried Lactobacillus acidophilus. Miner    Electrolyte Metab 1996; 22(1-3):92-6.-   34. Takayama et al., Bifidobacterium in gastro-resistant seamless    capsule reduces serum levels of indoxyl sulfate in patients on    hemodialysis. Am J Kidney Dis 2003; 41(3 Suppl 1):S142-5.-   35. Ranganathan et al., Probiotic amelioration of azotemia in 5/6th    nephrectomized Sprague-Dawley rats. ScientificWorldJournal 2005;    5:652-60.-   36. Ranganathan et al., Pilot study of probiotic dietary    supplementation for promoting healthy kidney function in patients    with chronic kidney disease. Adv Ther 2010; 27(9):634-47.-   37. Niwa and Ise, Indoxyl sulfate, a circulating uremic toxin,    stimulates the progression of glomerular sclerosis. J Lab Clin Med    1994; 124(1):96-104.

Although only a few exemplary embodiments have been described in detail,those skilled in the art will readily appreciate that many modificationsare possible in the exemplary embodiments without materially departingfrom the novel teachings and advantages of this disclosure. Accordingly,all such modifications and alternative are intended to be includedwithin the scope of the invention as defined in the following claims.Those skilled in the art should also realize that such modifications andequivalent constructions or methods do not depart from the spirit andscope of the present disclosure, and that they may make various changes,substitutions, and alterations herein without departing from the spiritand scope of the present disclosure.

What is claimed is:
 1. A method of treating a subject with symptoms ofkidney disease, progressive renal fibrosis, age-related decline inkidney function, and/or cardiovascular disease comprising: treating asubject with a gut TMA lyase inhibitor, wherein said subject hassymptoms of kidney disease, progressive renal fibrosis, age-relateddecline in kidney function, and/or cardiovascular disease; and whereinsaid gut TMA lyase inhibitor comprises fluoromethyl choline.
 2. Themethod of claim 1, wherein a sample from said subject is assayed todetermine levels of eGFR, eCrCl, Cystatin C, KIM1, urinealbumin/Creatinine ratio, trimethylamine N-oxide (TMAO), TMA, and/or aTMA-containing compound prior to and/or after said treating.
 3. Themethod of claim 2, wherein said subject is determined to have reducedlevels of eGFR and/or urine albumin/Creatinine ratio prior to saidtreating.
 4. The method of claim 2, wherein said subject is determinedto have elevated levels of Cystatin C, KIM1, or urine albumin/Creatinineratio prior to said treating.
 5. The method of claim 2, wherein saidsample is selected from whole blood, serum, plasma, urine, and saliva.6. The method of claim 2, wherein said subject is determined to haveelevated levels of said TMAO prior to said treating.
 7. The method ofclaim 2, wherein said subject is determined to have reduced levels ofsaid TMAO after said treating.
 8. The method of claim 1, wherein saidgut TMA lyase inhibitor consists of said fluoromethycholine.
 9. Themethod of claim 1, wherein said subject has cardiovascular disease. 10.The method of claim 1, wherein said subject has kidney disease.
 11. Themethod of claim 8, wherein said kidney disease comprises chronic kidneydisease.
 12. The method of claim 1, wherein said subject has progressiverenal fibrosis.
 13. The method of claim 1, wherein said subject hasage-related decline in kidney function.
 14. The method of claim 1,wherein said subject has elevated levels of TMAO.
 15. A compositioncomprising: an agent that reduces the production of trimethylamine (TMA)or trimethylamine-n-oxide (TMAO) in a subject, wherein said agentcomprises a halomethyl choline, and an enteric coating that provides forrelease of a majority of said agent in the colon and/or cecum of saidsubject.
 16. The composition of claim 15, wherein said halomethylcholine comprises fluoromethyl choline.